Techniques have been developed and media have been formulated that will support replicative cultures of monkey and human liver epithelial cells that exhibit some hepatocellular characteristics for 20 and 12 culture population doublings, respectively. However, monkey and transformed human liver epithelial cells established in these media will cease replicating after a few passages unless the media are supplemented with arginine. A similar observation was made using the human hepatoblastoma cell line Hep-G2. Thus, we speculate that hepatocytes may commonly lose portions of the urea cycle metabolic pathway as they undergo "retrograde differentiation" and/or transformation. In addition, we have found by measuring albumin and keratin species 18 and 19 that, initially, the transformed human liver epithelial (THLE) cells probably originate from hepatocytes or from cells that are of the hepatocyte lineage. However, with continued growth the THLE cells also co-exhibited liver ductal epithelial cell characteristics. Finally, preliminary investigations have demonstrated that inter-splenic injection protocols and "tissue equivalent" matrices may be efficacious for inducing replicative cultures of normal and transformed liver epithelial cells to re-express hepatocel- lular phenotypic expressions.